Tranilast prevents renal interstitial fibrosis by blocking mast cell infiltration in a rat model of diabetic kidney disease.

Renal interstitial fibrosis is a final pathway that is observed in various types of kidney diseases, including diabetic kidney disease (DKD). The present study investigated the effect of tranilast on renal interstitial fibrosis and the association between its role and mast cell infiltration in a rat model of DKD. A total of 30 healthy 6­week­old male Sprague­Dawley rats were randomly divided into the following four groups: Normal control group; DKD model group; low­dose tranilast group (200 mg/kg/day); and high­dose tranilast group (400 mg/kg/day). The morphological alterations of tubulointerstitial fibrosis were evaluated by Masson's trichrome staining, while mast cell infiltration into the renal tubular interstitium was measured by toluidine blue staining and complement C3a receptor 1 (C3aR) immunohistochemical staining (IHC). The expression of fibronectin (FN), collagen I (Col­I), stem cell factor (SCF) and proto­oncogene c­kit (c­kit) was detected by IHC, western blotting and reverse transcription­quantitative­polymerase chain reaction. The results demonstrated that tubulointerstitial fibrosis and mast cell infiltration were observed in DKD model rats, and this was improved dose­dependently in the tranilast treatment groups. The expression of FN, Col­I, SCF and c­kit mRNA and protein was upregulated in the tubulointerstitium of DKD model rats compared with the normal control rats, and tranilast inhibited the upregulated expression of these markers. Furthermore, the degree of SCF and c­kit expression demonstrated a significant positive correlation with C3aR­positive mast cells and the markers of renal interstitial fibrosis. The results of the present study indicate that mast cell infiltration may promote renal interstitial fibrosis via the SCF/c­kit signaling pathway. Tranilast may prevent renal interstitial fibrosis through inhibition of mast cell infiltration mediated through the SCF/c-kit signaling pathway.

Norcantharidin inhibits the expression of extracellular matrix and TGF-ß1 in HK-2 cells induced by high glucose independent of calcineurin signal pathway.

Norcantharidin (NCTD) was shown in our previous studies to attenuate renal tubulointerstitial fibrosis in rat models with diabetic nephropathy (DN). The aim of this study was to determine the effects of NCTD on the expression of extracellular matrix (ECM) and TGF-ß1 in HK-2 cells stimulated by high glucose and on calcineurin (CaN)/NFAT pathway. Whether or not the antifibrotic effect of NCTD on renal interstitium was dependent on its inhibition of CaN pathway was also investigated. Experimental concentrations of NCTD were verified by cytotoxic test and MTT assay. HK-2 cells were transfected with CaN small interference RNA (siRNA). The mRNA and protein expressions of FN, ColIV, TGF-ß1, and CaN in HK-2 cells were detected by real-time PCR and western blot. The CaN/NFAT pathway was examined by indirect immunofluorescence and western blot. Our study revealed that NCTD concentrations over 5 mg/l had overt cytotoxicity on HK-2 cells. Meanwhile, both 2.5 and 5 mg/l NCTD inhibited HK-2 cell proliferation (P < 0.05). NCTD inhibited the upregulation of FN, ColIV, and TGF-ß1 of HK-2 cells stimulated by high glucose (P < 0.05), and also significantly downregulated the expression of CaN mRNA and protein in HK-2 cells (P < 0.05). In addition, not only was the nuclear translocation of NFATc inhibited, but its protein level in the nucleus was also reduced. Following CaN siRNA transfection, CaN mRNA and protein expression were significantly decreased. In contrast, the protein levels of FN, ColIV, and TGF-ß1 increased in HK-2 cells stimulated by high glucose (P < 0.05). However, NCTD treatment downregulated their expression. These results indicated that NCTD could decrease the expression of ECM and TGF-ß1 in HK-2 cells stimulated by high glucose, downregulate CaN expression, and block the CaN/NFAT signaling pathway. However, the effect of NCTD on inhibition of the expression of ECM and TGF-ß1 was not associated with its inhibition of the CaN/NFAT pathway.

Norcantharidin inhibits proliferation and fibronectin expression of HK-2 cells induced by albumin in vitro.

The present study aims to observe the effects of NCTD (norcantharidin) on proliferation and FN (fibronectin) expression in human renal proximal tubular epithelial cell line (HK-2) induced by albumin in vitro. HK-2 cells were divided into control group, albumin group and different concentration of NCTD intervention groups. Proliferation of HK-2 cells was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], FN protein in culture media of HK-2 cells was examined by ELISA, and FN mRNA was analysed by RT-PCR (reverse transcription-PCR). We chose less than 5.0 mg/l of NCTD as the experimental concentration for the cytotoxicity test. MTT score was higher in the albumin group than in the control group (P<0.05). As compared with that of the albumin group, MTT score and FN protein concentration decreased, FN mRNA significantly down-regulated in NCTD intervention groups respectively (P<0.05). Our study showed that NCTD could inhibit the albumin-induced cell proliferation and FN expression in HK-2 cells, which might further prove the anti-fibrotic role of NCTD in proteinuria-associated tubulointerstitial damage.

Norcantharidin attenuates tubulointerstitial fibrosis in rat models with diabetic nephropathy.

OBJECTIVE: To investigate the effects of norcantharidin (NCTD) on tubulointerstitial fibrosis of diabetic nephropathy (DN) in streptozotocin-induced rat model. METHODS: Sprague-Dawley rats were randomly divided into control group, model group, low-dose NCTD (0.05 mg/kg/day) group, and high-dose NCTD (0.1 mg/kg/day) group. The model group was induced by injection intraperitoneally with 30 mg/kg streptozotocin in 0.1 mol/L sodium citrate solution (pH 4.5), after high-calorie foods were given for 2 months. NCTD was administered daily after the DN rat model was built. Rats were sacrificed at the end of the third and the eighth week; renal fibrosis and the expression of FN, collagen IV, TGF-ß1, and calcineurin (CaN) were detected by Masson and immunohistochemistry staining, respectively. RESULTS: Tubulointerstitial fibrosis was observed in DN rats, this kind of pathological changes was ameliorated in NCTD treatment group (p < 0.05). The expressions of FN, collagen IV, and TGF-ß1 protein increased in the tubulointerstitial field of DN rats compared with the rats in control group. NCTD treatment could dose-dependently decrease their expression and reverse the fibrotic degree (p < 0.05). Meanwhile, the expression of CaN was detected in tubular fields of normal kidney and increased in the tubulointerstitial field in DN rats. However, NCTD downregulated its expression in a dose-dependent manner (p < 0.05). CONCLUSIONS: NCTD could downregulate FN, collagen IV, and TGF-ß1 expression in tubulointerstitial fields and attenuate tubulointerstitial fibrosis in the early stage of DN rats. NCTD also alleviated the expression of CaN in tubules in DN. The relationship between the role of NCTD's anti-tubulointerstitial fibrosis and its inhibition to CaN expression remains to be further elucidated.

Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas.

BACKGROUND: p75NTR has been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75NTR in esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75NTR+ cells. METHODS: p75NTR expression in ESCC was assessed by immunohistochemistry. p75NTR+ and p75NTR- cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75NTR+ and p75NTR- cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, 64copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75NTR+ cells. RESULTS: In ESCC specimens, p75NTR was found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75NTR+ cells was 1.6%-3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75NTR+ cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75NTR+ cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75NTR+ cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, beta1-integrin and beta4-integrin, were lower in p75NTR+ cells. In addition, p75NTR+ cells generated both p75NTR+ and p75NTR- cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75NTR+ cells were found to be more resistant to DDP and exhibited lower 64copper accumulation than p75NTR- cells. CONCLUSION: Our results demonstrated that p75NTR+ cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75NTR+ cells may probably be attributable to decreased expression of CTR1.